Pai-1 determination and use thereof

ABSTRACT

A monoclonal antibody which binds a human endothelial type plasminogen activator inhibitor (PAI-1) produced by dexamethasone-treated human HT-1080 fibrosarcoma cells may be used, inter alia, for determining PAI-1 protein abundance in tumor tissue or a sample of a body fluid. Measurements of this parameter may be useful in predicting the presence or metastasis of a tumor, or of predicting the progression of a known malignant tumor.

[0001] The present invention is a continuation-in-part application ofapplication Ser. No. 900,364 filed Jun. 18, 1992, which is a Rule 1.62continuation application of Ser. No. 752,990 filed Sep. 3, 1991, whichis a Rule 1.62 continuation application of Ser. No. 035,995 filed Mar,11, 1987.

TECHNICAL FIELD

[0002] This invention relates to monoclonal antibodies, a method ofproducing such antibodies, hybridoma cells capable of producing theantibodies and uses of the antibodies. Furthermore, the inventionrelates to the prognostic and diagnostic use of PAI-1 determinations ine.g. plasma samples, and to measurement of uPA:PAI-1 complexes and usesthereof.

BACKGROUND ART

[0003] The fusion of mouse myeloma cells with spleen cells fromimmunized mice (Köhler and Milstein, Nature (1975), 256, 496-497) wasthe first indication that it is possible to obtain continuous cell lineswhich produce homogenous (so-called “monoclonal”) antibodies. Sincethen, a large number of attempts have been made to produce varioushybrid cells (so-called “hybridomas”) and to employ the antibodiesformed by these cells for various scientific investigations (cf. CurrentTopics in Microbiology and Immunology, volume 81—“LymphocyteHybridomas”, F. Melchers et al., Springer-Verlag (1978) and referencestherein; C. J. Barnstable et al., Cell, (1978), 14, 9-20; P. Parham., W.F. Bodmer, Nature (1978), 276, 397-399; Handbook of ExperimentalImmunology, 3rd edition, vol. 2, D. M. Wier, editor, Blackwell, 1978,Chapter 25, Chem. Eng. News, 15-17 (1979); Kennett, R. H., McKearn, J.T., and Bechtol, K. B. (1980) Monoclonal Antibodies. Hybridomas: A NewDimension in Biological Analysis (Plenum, New York)). These reportsdescribe the principal techniques for the production of monoclonalantibodies by hybridomas.

[0004] Monoclonal antibodies against human plasminogen activators(urokinase-type (u-PA) and tissue-type (i-PA)) and produced byhybridomas have been prepared and have been used for purification,identification, and immunochemical localization of the activators andtheir proenzymes (Kaltoft, K., Nielsen, L. S., Zeuthen, J., and Danø, K.(1982) Proc. Natl. Acad. Sci. USA, 79, 3720-3723; Nielsen, L. S.,Hansen, J. G., Andreasen, P. A., Skriver, L., Danø, K., and Zeuthen, J.(1983) The EMBO Journal, 2, 115-119; Nielsen, L. S., Hansen, J. G.,Skriver, L., Wilson, E. L., Kaltoft, K., Zeuthen, J., and Danø, K.(1982), Biochemistry, 21, 6410-6415; Danø, K., Dabelsteen, E., Nielsen,L. S., Kaltoft, K., Wilson, E. L., and Zeuthen, J. (1982), J. Histochem.Cytochem., 30, 1165-1170). Andreasen, P. A., Nielsen, L. S.,Grøndahl-Hansen, J., Skriver, L., Zeuthen, J., Stephens, R. W., andDanø, K. (1984), The EMBO Journal, 3, 51-56). It has recently been shownthat inhibitors of plasminogen activators play an important role in theregulation of the plasmin mediated proteolysis. Such inhibitors havebeen identified in a variety of tissues, body fluids and cultured celllines (Holmberg, L, Lecander, I., Persson, B., and {dot over (A)}stedt,B. (1978), Biochim. Biophys. Acta, 544 128-137; Seifert, S. C. andGelehrter, T. D. (1978) Proc. Natl. Acad. Sci. USA, 75, 6130-6133;Chmielewska, J., Ränby, M., and Wiman, B. (1983) Thromb. Res., 31,427-431; Emeis, J. J., Van Hindsbergh, V. W. M., Verheijen, J. H. andWijngaards, G. (1983) Biochem. Biophys. Res. Commun., 110, 391-398;Golder, J. P. and Stephens, R. W. (1983) Eur. J. Biochem., 136, 517-522;Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D.(1983), Proc. Natl. Acad. Sci. USA, 80, 2956-2960; Philips, M., Juul,A.-G., and Thorsen, S. (1984) Biochim. Biophys. Acta, 802, 99-110;Vassalli, J.-D., Dayer, J.-M., Wohlwend, A. and Belin, D. (1984) J. Exp.Med., 159, 1653-1668; Erickson, L. A., Ginsberg, M. H., and Loskutoff,D. J. (1984), J. Clin. Invest., 74, 1465-1472; Cwikel, B. J.,Barouski-Miller, P. A., Coleman, P. L., and Gelehrter, T. D. (1984), J.Biol. Chem., 259, 6847-6851; {dot over (A)}stedt, B., Lecanders, I.,Brodin, T., Lundblad, A., and Löw, K. (1985), Thrombos. Haemost., 53,122-125; J. Biol. Chem. (1985), 260, 7029-7034) .

[0005] The mutual relationship of these inhibitors is at present notfully clarified, although recent evidence indicates that at least threeimmunologically dissimilar types of plasminogen activitor inhibitorsexist. These include (1) protease nexin, (2) plasminogen activatorinhibitor purified from placenta ({dot over (A)}stedt, B., Lecander, I.,Brodin, T., Lundblad, A., and Löw, K., (1985) Thromb. Haemost. 53,122-125), and (3) plasminogen activator inhibitors that inhibit u-PA andt-PA and which typically have been obtained from human endothelialcells, human fibrosarcoma cells (HT-1080), human blood platelets, andrat hepatoma cells (HTC), in the following referred to as endothelialtype plasminogen activator inhibitor (e-PAI).

[0006] An inhibitor with remarkable similarities to e-PAI has been foundin human plasma (Thorsen, S. and Philips, M. (1984) Biochim. Biophys.Acta 802, 111-118).

[0007] Monoclonal antibodies against placental plasminogen activatorinhibitor have been prepared and such antibodies have been used for thepurification of said inhibitor ({dot over (A)}stedt, B., Lecander, I.,Brodin, T., Lundblad, A., and Löw, K., (1985) Thromb. Haemost. 53,122-125).

DISCLOSURE OF INVENTION

[0008] The present invention provides monoclonal antibodies against theendothelial type plasminogen activator inhibitor and immunologicallysimilar inhibitors.

[0009] The term “immunologically similar inhibitors” denotes plasminogenactivator inhibitors which cross-react with polyclonal or monoclonalantibodies raised against inhibitors derived from any of the sourcesmentioned in connection with the above definition of endothelial typeplasminogen activator inhibitor.

[0010] The provision of these antibodies makes it possible to study therole of plasminogen activator inhibitors in plasmin mediated proteolysisincluding fibrinolysis and the mutual relationship of the abovementioned plasminogen activator inhibitors. Moreover, such monoclonalantibodies are useful for the purification of plasminogen activatorinhibitor by means of immunoadsorption chromatography, for removal ofthe inhibitor from body fluids and other biological materials by meansof immunoadsorption, for neutralization of the inhibitory activity ofthe plasminogen activator inhibitor and for the detection,identification and quantification, e.g. by the ELISA technique, ofplasminogen activator inhibitor in body fluids, normal or malignantcells and tissues, and other biological materials.

[0011] The invention also provides a method of producing the abovementioned antibodies. This method comprises fusing myeloma cells withantibody-producing cells obtained from mammals which have been immunizedwith endothelial type plasminogen activator inhibitor or immunologicallysimilar inhibitors or with antibody-producing cells which in vitro hasbeen immunized with said plasminogen activator inhibitor, and selectingthe hybridomas producing antibodies against the above mentionedinhibitors. Thus the hybridomas are produced by a derivation of themethod of Köhler and Milstein mentioned above. The antibody-producingcells used are preferably spleen cells or lymph node cells. Theparticular species of mammals from which the myeloma andantibody-producing cells are derived is not critical insofar as it ispossible to fuse the cells of the one species with another, e.g. mouseto rat, rat to human, or mouse to human.

[0012] It is preferred, however, to use the same species of animal as asource of both myeloma and anti plasminogen activator inhibitorantibody-producing cells. One preferred cell line for the practice ofthis invention is a fused cell hybrid between a plasminogen activatorinhibitor primed mouse spleen cell and a mouse myeloma cell.

[0013] The hybridomas resulting from the fusion are systematicallyexamined for production of antibodies which selectively react withplasminogen activator inhibitor.

[0014] It should be noted that monoclonal antibodies raised against asingle antigen may be distinct from each other depending on thedeterminant that induced their formation; but for any given hybridoma(clone), all of the antibodies it produces are monospecific for aparticular antigenic determinant in the plasminogen activator inhibitormolecule.

[0015] The invention also relates to hybridoma cells capable ofproducing monoclonal antibodies against the endothelial type plasminogenactivator inhibitor and immunologically similar inhibitors.

[0016] In general, the production of the hybridomas comprises thefollowing steps:

[0017] A. Immunization of mammals with partially purified plasminogenactivator inhibitor. Balb/c-mice have been found useful for thispurpose, but other mammals can also be used. The immunization scheme andthe concentration of plasminogen activator inhibitor should be selectedSuch that adequate amounts of antigen-stimulated lymphocytes are formed.

[0018] B. Obtaining the spleens or lymph nodes of the immunized mammalsand preparation of a spleen cell suspension or a lymph node cellsuspension in a suitable medium.

[0019] C. Fusion of the suspended spleen cells or lymph node cells withmyeloma cells of a suitable cell line (for example NS1-Ag 4/1 myelomacells), using a suitable fusion promotor (for example polyethyleneglycol). A ratio of about 10 spleen cells or lymph cells per myelomacell is preferred. A total volume of about 1 ml of fusion medium isadequate for ₁₀ ⁸ spleen cells or lymph node cells. The myeloma cellline used should preferably be of the so-called “drug resistant” type,so that, in a selective medium, unfused myeloma cells die whilst hybridssurvive. Cell lines resistant to 8-azaguanine, which cells lack theenzyme hypoxanthine-guanine phosphoribosyl transferase and whichtherefore cannot grow in a HAT medium (hypoxanthine, aminopterin,thymidine), are most frequently used.

[0020] The myeloma cell line used should also preferably be of the“non-secreting” type so that it does not itself form antibodies or H orL chains of immunoglobulins.

[0021] D. Dilution and cultivation in individual vessels of the unusedspleen cells or lymph node cells, the unfused myeloma cells and thefused cells in a selective medium, in which the unfused myeloma cells donot divide so that the unfused cells die (about 1-2 weeks). Theindividual fused cells are isolated by adjusting the volume of thediluent so that a given number of cells (about 1-4) is placed in eachindividual vessel (for example each well of a microtitre plate). Themedium (for example HAT medium) prevents the growth of the resistant(for example against 8-azaguanine) unfused myeloma cell line, and thusit dies. The unfused spleen cells or lymph node cells have only alimited number of division cycles and hence these cells also die after acertain period (about 1-2 weeks) In contrast, the fused cells continueto divide since they have inherited permanent growth from the parentmyeloma cells and the ability to synthesize the enzymehypoxanthine-guanine phosphoribosyltransferase from the parent spleencells or lymph node cells, and thus they are able to survive in theselective medium.

[0022] E. Checking for the presence of antibodies against plasminogenactivator inhibitor in each vessel.

[0023] F. Selecting (for example by limiting dilution) and cloning thehybridomas which produce the desired antibody.

[0024] When the desired hybridoma has been selected and clonedmonoclonal antibodies of very high purity are obtained when thehybridomas are cultured in a suitable medium for a certain time and theantibody is obtained and purified from the supernatent. A suitablemedium and the optimum culture time can easily be determined. This invitro technique provides monoclonal antibodies which are contaminatedwith only small amounts of proteins from the heterologous serum (forexample fetal calf serum).

[0025] In order to produce a significantly higher concentration ofmonoclonal antibodies of only very slightly reduced purity, the selectedhybridoma can be injected into a, preferably syngeneic or semisyngeneic,mouse. After a certain incubation time, this leads to the formation of atumour in the mouse which releases high concentrations of antibody (5-20mg/ml) in the blood and in the peritoneal exudate (ascites) of the hostanimal. Even though these mice have normal antibodies in the blood andascites, nevertheless these only arise at a concentration of about 5% ofthe monoclonal antibodies.

[0026] According to the nomenclature recommended by the Subcommittee onFibrinolysis of the International Committee on Thrombosis andHemostasis, Jun. 8, 1986, endothelial type plasminogen activatorinhibitor (e-PAI) should be denominated plasminogen activator inhibitortype 1 (PAI-1). In the following description and Examples 6-9, thisnomenclature has been used.

[0027] Prognostic significance of PAI-1 in extracts from human tumours.

[0028] Since it is well established that proteolytic activity isnecessary for tumour cell spreading, molecules involved in theregulation of invasion and metastasis are attractive asprognostic/diagnostic tools. The challenge is to identify those patientsat low risk and those at high risk of recurrence. An accurate means ofdistinguishing those at high or low risk of relapse would spare patientsat low risk from severe side effects of adjuvant chemotherapy while highrisk patients could be offered intensive chemotherapy.

[0029] Breast cancer

[0030] Foucre et al. (1991) Br. J. Cancer 64, 926-932, found a 74-foldincrease of PAI-1 in breast tumours as compared with normal breasttissue. Similar findings were reported by Sumiyoshi et al., 1991 whoalso found that increased levels of tumour PAI-1 were directlyproportional to the number of tumour positive axillary lymph nodes.

[0031] Jänicke et al. (1991) Sem. Throm. Hemostasis 17, 303-312, werethe first to describe the prognostic role of PAI-1 in breast tumourextracts. Including tumour extracts from 113 breast cancer patients,high tumour PAI-1 content as determined by a sandwich ELISA, was shownto be an independent and significant predictor of poor prognosis. Twolater studies by Grøndahl-Hansen et al. (1993) Cancer Res. 53,2513-2521, including 190 patients and by Foekens et al. (1994) J. Clin.Oncol. 12, 1648-1658, including 657 patients, confirmed the prognosticimpact of PAI-1 in patients with node negative and node positive breastcancer.

[0032] Of particular interest is that PAI-1 seems to be an independentprognostic variable, i.e. measurement of tumour PAI-1 contentcontributes significantly to the prognostic information which can beobtained by other prognostic parameters. For example, in the subgroup ofpatients with estrogen receptor positive tumours, who have a betterprognosis than patients with estrogen receptor negative tumours, PAI-1tumour measurements allows for a further prognostic stratification(Grøndahl-Hansen et al., supra). Also in the subgroup of patients with 1to 3 tumour positive axillary lymph nodes, PAI-1 could be used toseparate the patients into significantly different prognostic groups(Grøndahl-Hansen et al., supra). This latter observation suggests thatin the group of patients with 1-3 tumour positive lymph nodes a subgroupof high-risk patients can be identified and these women might be offeredmore intensive chemotherapy.

[0033] In the study by Foekens et al., supra, PAI-1 appeared to be thestrongest biochemical prognostic marker, when uPA, cathepsin D, pS2,estrogen and progesterone receptors were included as the otherbiochemical variables, indicating the importance of PAI-1 measurementsin predicting prognosis in breast cancer.

[0034] Gastric cancer

[0035] Analyzing PAI-1 tumour content in 76 patients with completeresection of their gastric cancer, Nekarda et al. (1994) Cancer Res. 54,2900-2907, were able to demonstrate prognostic significance of PAI-1,high PAI-1 being significantly associated with poor prognosis when usingthe best cut-off value to part the patients in two groups, 45 patientshaving low values and 31 having high. In a multivariate Cox regressionanalysis, PAI-1 was proven to be an independent prognostic factor withnodal status and WHO-classification as the two only other prognosticfactors.

[0036] Pancreatic cancer

[0037] Applying immunohistochemistry on paraffine sections, Takeuchi etal. (1993) Am. J. Gastroenterology 68, 1928-1933, studied the prognosticrole of tumour PAI-1 and PAI-2 staining intensity in 97 patients withpancreatic cancer. While strong staining intensity for PAI-2 wassignificantly associated with long overall survival, PAI-1 stainingintensity had no impact on survival.

[0038] Colon cancer

[0039] Tumour PAI-1 levels as measured by ELISA, are found significantlyelevated in primary colon adenocarcinomas and their metastasis ascompared to normal colon mucosa: normal mucosa<primary tumour<livermetastasis (Sier et al. [1994] Gastroenterology 107, 1449-1456). Theauthors conclude that the high PAI-1 content in colorectal cancermetastasis in the liver is associated with an inactivation of theenhanced urokinase cascade, which might allow tumour cells to settle inthe liver. Ganesh et al. (1994) Cancer Res. 54, 4065-4071, studied theprognostic impact of PAI-1 in 92 colon carcinomas and found nosignificant correlation between PAI-1 as determined by sandwich ELISA,and patient outcome.

[0040] In a recent study performed at the Finsen Laboratory(unpublished), PAI-1 content was investigated by ELISA in normal colonmucosa, in the periphery of colon adenocarcinomas and in the center ofthe tumours. Normal mucosa had approximately 10 fold less PAI-1 than theperiphery of the tumour, while the center of the tumour hadapproximately 50 fold higher PAI-1 levels than the normal mucosa. Whencomparing tumour PAI-1 levels with clinical outcome in the relative lownumber of patients, a trend towards statistical significance in survivalbetween high versus low PAI-1 was seen (FIG. 6)

[0041] Lung cancer

[0042] In a retrospective study including tumour tissue from 106 lungadenocarcinoma patients we determined PAI-1 by sandwich ELISA. Using theupper and lower quartiles as cut-off points, high PAI-1 was shown to besignificantly (P=0.017) correlated with short overall survival (Pedersenet al. [1994] Cancer Res. 54, 120-123). In Cox multivariate analysis,including clinical parameters and tumour uPA, PAI-1 was shown to be anindependent prognostic marker for survival, stage being the only othersignificant prognostic factor. When analyzing the 69 stage 1 patientsseparately and using the median as cutoff point, high levels of PAI-1were significantly (P=0.038) associated with poor prognosis.

[0043] In a second retrospective study including tumour tissue from 84patients with squamous cell lung cancer and 38 patients with large celllung cancer (Pedersen et al. [1994], Cancer Res. 54, 4671-4675), therewas a non-significant trend towards high PAI-1 levels being associatedwith poor prognosis in squamous cell lung cancer. However, combininghigh tumour levels of PAI-1 and high tumour levels of urokinase typeplasminogen receptor (27 of the 84 patients), a highly significant(P=0.008) association with short survival was seen. PAI-1 did not haveany significant correlation with survival in the group of large celllung cancer patients (Pedersen et al., supra).

[0044] Ovarian cancer

[0045] A number of studies have shown that tumour concentration of PAI-1in ovarian cancers is significantly higher as compared with benignovarian tissue specimens (Casslén et al. [1994) Eur. J. Cancer,1302-1309; Kuhn et al. [1995] Gy. Oncol., in press).

[0046] In a recent study by Kuhn et al., supra, PAI-1 as determined byELISA was shown to predict survival in advanced ovarian cancer patientsafter radical surgery and platinum-based chemotherapy, i.e. high tumourlevels of PAI-1 was significantly (P=0.01) associated with shortsurvival. A best cutoff point was defined dividing the patients into 27with low level and 24 with high PAI-1 tumour levels. In the multivariateanalysis residual tumour after operation and high PAI-1 or high uPA werethe only prognostic factors.

[0047] Plasma PAI-1

[0048] All studies published until now on the prognostic value of PAI-1are based on determinations in tumour extracts. For most types ofcancer, the development of more effective diagnostic methods hasresulted in earlier detection and thus smaller tumour size at the timeof surgery. This is making it increasingly difficult to acquire accessto frozen, unfixed tumour samples. Therefore, the conversion of thePAI-1 assay from a tumour extract based test to one that can analyzeplasma samples, would significantly increase its clinical use. Samplecollection would be much easier and less invasive.

[0049] PAI-1 can be detected in plasma and has been shown to be elevatedin patients with pancreatic cancer (Sandberg et al. (1992] 69,2884-2887), ovarian cancer (Casslén et al., supra) and in urinary tractcancers (Bashar et al. [1994] Urol. Int. 52, 4-8). In the last study,plasma levels were significantly higher in a group of patients withmetastatic disease than in patients without distant metastasis. Anassociation between the degree of cancer cell atypia and plasma PAI-1levels was reported.

[0050] In a recent study performed at the Finsen Laboratory(unpublished), it was found that patients with colon adenocarcinomaspreoperatively had increased plasma PAI-1 levels as compared to healthycontrol individuals. A correlation was found between tumour and plasmaPAI-1 content in the individual patients, and subsequent correlation tosurvival showed significant different survival among patients with lowversus high plasma PAI-1 content (FIG. 7).

[0051] In breast cancer patients, we have also recently been able toshow an increased plasma PAI-1 level in the cancer patients as comparedto healthy controls.

[0052] Diagnostic significance of plasma PAI-1 in human cancer

[0053] Follow-up of patients in either surgically or medically inducedremission most often involves only clinical examination. With the highrecurrence rate in many cancer types, a sensitive diagnostic assay, e.g.an assay as outlined in Example 6, which is capable of identifyingnon-clinically evident recurrence will be of significant value.

[0054] Colon cancer

[0055] It has recently been observed that in patients with advancedcolon adenocarcinomas (Dukes D) plasma PAI-1 levels are correlated withtumour burden, e.g. high plasma PAI-1 level before debulking surgery,significant fall postoperatively and then steady increase during diseaseprogression (FIG. 8) (unpublished data).

[0056] In its broadest aspect, the invention relates to a method ofpredicting the presence or progression of a malignant tumour in asubject having or suspected of having a malignant tumour, the methodcomprising

[0057] (a) determining at a first point in time (I) one or more of PAI-1DNA abundance, PAI-1 mRNA abundance, or PAI-1 protein abundance intumour tissue or a sample of a body fluid such as plasma, serum or urinefrom said subject,

[0058] (b) determining at a later point in time (II) one or more ofPAI-1 DNA abundance, PAI-1 mRNA abundance, or PAI-1 protein abundance ina sample of body fluid from said subject,

[0059] (c) determining the difference between the abundance of saidPAI-1 DNA, PAI-1 mRNA, or PAI-1 protein determined at said first pointin time (I) with the value determined at said later point in time (XI),and

[0060] (d) correlating said difference with an established level ofdifference which is indicative of a high likelihood of tumour presenceor metastasis.

[0061] In particular, the invention relates to a method wherein saidfirst point in time (I) is preoperatively, and said second point in time(II) is at least 2 weeks postoperatively, such as 4, 6 or 8 weeks oreven 3, 6, 12, 18 or 24 months post-operatively. It is contemplated thatif the difference between the abundance of said PAI-1 DNA, PAI-1 mRNA,or PAI-1 protein determined at said first point in time (I) and thevalue determined at said later point in time (II) is more than 50% ofthe value determined at the first point in time (I), such as 75% or 100%or more, this is indicative of a non-clinically (and possibly alsoclinically) evident recurrence.

[0062] In a specific embodiment, the method comprises the steps of:

[0063] (a) testing a tissue section from a malignant tumour or a sampleof a body fluid from a patient suspected of having a malignant tumour,said sample taken at a first point in time (I), with an antibody reagentspecific for PAI-1 protein under antibody binding conditions,

[0064] (b) determining the binding of the reagent to PAI-1 protein insaid tissue section or sample of a body fluid taken at said first pointin time (I),

[0065] (c) testing a sample of a body fluid from a patient suspected ofhaving a malignant tumour, said sample taken at a later point in time(II), with an antibody reagent specific for PAI-1 protein under antibodybinding conditions,

[0066] (d) determining the binding of the reagent to PAI-1 protein insaid sample of body fluid taken at said later point in time (II),

[0067] (e) determining the difference between the level of said PAI-1protein determined at said first point in time (I) with the valuedetermined at said later point in time (II), and

[0068] (f) correlating said difference with an established level ofdifference which is indicative of a high likelihood of tumour presenceor metastasis.

[0069] By the term “antibody reagents” is meant whole antibodies andparts thereof, either alone or conjugated with other moieties.Antibodies include polyclonal antibodies, monoclonal antibodies, andsingle chain antibodies. Antibody fragments are those that bind thePAI-1 protein, including Fab and F(ab)₂ fragments, inter alia. Theantibody reagents may comprise antibodies made in animals or byrecombinant DNA techniques. Also the antibody reagents include antibodyand antibody fragments conjugated to, among other moieties, detectablelabels, such as enzymatic labels and fluorescent labels. Other usefullabels which may be comprised by the antibody reagents includeradionucleotides.

[0070] Antibody binding conditions are generally well known in the artand, for the most part, will include neutral pH, moderate salt,temperatures between 2-3° C. and 37° C., incubation times betweenseveral minutes and overnight or longer. Preferred conditions includethose described in Examples 6-9.

[0071] It will be readily appreciated by those of ordinary skill in theart that the details of an antibody binding procedure may be adjusted tofavour improved signal to noise ratios or sensitivity, rapidity orcompleteness, and the like. Moreover, conditions may be adjusted toaccomodate different histological procedures for fixation and staining.Finally, it will be appreciated that titers and appropriate dilutionswill be different for different antibody reagent preparations.

[0072] In a presently preferred embodiment of the above method, saidantibody reagent is an antibody according to the invention and thedetermination of the PAI-1 protein level is performed by using animmunoassay, such as an ELISA or RIA, or by using an activity assay.

[0073] In yet another embodiment of the present invention, the abundanceof PAI-1 mRNA or PAI-1 DNA in a tumour tissue sample may be detected byis situ hybridization using PAI-1 sequence specific probes, or byhybridization of PAI-1 sequence specific probes to mRNA or DNA fromnormal and tumour tissue.

[0074] In a still further embodiment of the present invention, thepolymerase chain reaction (“PCR”) is used to detect PAI-1 DNA or mRNA ina tumour tissue sample.

[0075] In another embodiment, RNA (“Northern”) blotting may be used inthe methods of the invention. According to this method, RNA is isolatedfrom tumour tissue by any of a number of standard procedures.

[0076] Predictive value of tumour PAI-1 in human cancer

[0077] As shown in Example 6, a proportion of colon adenocarcinomacancer patients have high levels of plasma PAI-1 which predict a poorprognosis. Accordingly, patients with high PAI-1 levels will becandidates for anti-PAI-1 therapy treatment. Example 7 describes apredictive assay to identify patients who will potentially benefit fromsuch a treatment.

[0078] The invention further relates to a method of predicting theprognosis of an individual subject having or suspected of having amalignant tumour, the method comprising

[0079] a) determining the level of PAI-1 DNA abundance, PAI-1 mRNAabundance, or PAI-1 protein abundance in malignant or potentiallymalignant tissue or another sample, such as plasma, serum, or urine,from a number of subjects having or suspected of having a malignanttumour,

[0080] b) establishing a threshold level of PAI-1 DNA, PAI-1 mRNA orPAI-1 protein above or equal to which a value is indicative of a highlikelihood of non-clinically evident tumour metastasis resulting in apoor prognosis,

[0081] c) correlating the level of PAI-1 DNA, PAI-1 mRNA or PAI-1protein of the individual subject with the value established in b) inorder to determine the prognosis of the individual subject, andoptionally

[0082] d) if the likelihood of a poor prognosis is high, allocating theindividual subject to subsequent antineoplastic treatment.

[0083] Whether a prognosis is good or poor depends upon the likelihoodof metastasis. A “high likelihood” of tumour metastasis means that thereis more than 40% risk of tumour metastasis within about five years.

[0084] Another aspect of the invention relates to a method of selectinga subject having or suspected of having a malignant tumour foranti-PAI-1 treatment, the method comprising

[0085] a) determining the level of PAI-1 DNA abundance, PAI-1 mRNAabundance, or PAI-1 protein abundance in malignant or potentiallymalignant tissue or another sample, such as plasma, serum, or urine,from the subject,

[0086] b) correlating said value with an established threshold leveldetermined as described above, and

[0087] c) selecting for anti-PAI-1 treatment patients having a PAI-1level above or equal to the pre-determined threshold level.

[0088] The above diagnostic, prognostic and predictive methods may beapplied to samples from any subject having or suspected of having amalignant tumour, e.g. to samples from a subject or patient who has beenestablished to have a carcinoma in situ.

[0089] In certain embodiments of the above methods, the subject orpatient is a patient who has been established to have a high risk ofdeveloping a malignant tumour by having a high-risk-indicating score ofa tumour marker such as a serum/plasma tumour marker or by having a geneor gene product which indicates that the patient is at high risk ofdeveloping a malignant tumour; the malignant tumour being selected fromthe group consisting of mammary carcinomas, urological carcinomas e.g.prostate carcinoma and bladder carcinoma, gynaecological carcinomas e.g.ovarian carcinoma and cervical carcinoma, non-small cell lung tumours,gastrointestinal cancers, e.g. colon adenocarcinoma, and gastriccancers, brain tumours, sarcomas, haematological malignancy e.g.lymphoma and skin cancers e.g. melanoma and squamous cell skin cancer.

[0090] Prognostic value of uPA:PAI-1 complexes in patients with breastcancer

[0091] Tumour content of uPA, PAI-1 and uPAR has been shown to predictprognosis in breast cancer (Foekens et al., Foekens et al.,Grøndahl-Hansen et al., Grøndahl-Hansen et al.). These studies have beenbased on measurement of total amounts of uPA, uPAR or PAI-1. It is wellknown, however, that both uPA and PAI-1 can exist in active and inactiveforms (pro-uPA, uPA, latent PAI-1, active PAI-1). Active PAI-1, which ispresent in surplus in breast cancer tissue (Jänicke et al.), will formcomplexes with uPA but not with pro-uPA (Andreasen et al.). TheuPA:PAI-1 complex can bind uPAR and is internalized when uPAR bound(Nykjaer et al.). Tumour content of (active) uPA which might be anindicator of active proteolysis per se in the tumour tissue constitutesonly a small fraction of the total amount of uPA (Skriver et al.). Thereare at present no ELISA methods for a selective determination of(active) uPA. Since active PAI-1 only forms complexes with (active) uPAand not with pro-uPA, the amount of uPA:PAI-1 complexes could representan indirect measure of active uPA in a tissue and thereby a measure ofactive proteolysis.

[0092] Based on monoclonal and polyclonal uPA and PAI-1 antibodies, anELISA which with high sensitivity detects complexes between uPA andPAI-1, cf. Example 8 (FIG. 9) and a standard material consisting of invitro formed uPA:PAI-1 complexes have been developed.

[0093] A sensitive and specific uPA:PAI-1 complex ELISA and a stableuPA:PAI-1 complex standard preparation have been developed as describedin Example 8. By measuring complexes in breast cancer cytosols,immunoreactivity was found in all samples with large variations betweenthe samples (FIG. 10). A number of validation studies on the ELISA arecurrently being performed, including the use of different tumourextraction procedures. Upon completion of these studies, a breast cancercytosol bank will be used to determine the exact prognostic value ofuPA:PAI-1 complexes.

[0094] Similarly, the prognostic value of uPA:PAI-1 complexes is testedin extracts from other cancer types, e.g. colon, non-small cell lungcancer, gastric cancer, ovarian cancer, and cervical cancer. Also, thepresence of uPA:PAI-1 complexes in plasma, serum, and urine from cancerpatients is examined for a prognostic value in a similar manner as theexperiments described in Examples 6 and 7.

[0095] A further aspect of the invention thus relates to a method ofpredicting the presence or progression of a malignant tumour in asubject having or suspected of having a malignant tumour, the methodcomprising

[0096] (a) determining at a first point in time (I) uPA:PAI-1 complexesin tumour tissue or a sample of a body fluid such as plasma, serum orurine from said subject,

[0097] (b) determining at a later point in time (II) uPA:PAI-1 complexesin a sample of body fluid from said subject,

[0098] (c) determining the difference between the abundance of saiduPA:PAI-1 complexes determined at said first point in time (I) with theabundance determined at said later point in time (II), and

[0099] (d) correlating said difference with an established level ofdifference which is indicative of a high likelihood of tumour presenceor metastasis.

[0100] The method will generally comprise the following step:

[0101] (a) testing a tissue section from a malignant tumour or a sampleof a body fluid from a patient suspected of having a malignant tumour,said sample taken at a first point in time (I), with an antibody reagentspecific for uPA:PAI-1 complexes under antibody binding conditions,

[0102] (b) determining the binding of the reagent to uPA:PAI-1 complexesin said tissue section or sample of a body fluid taken at said firstpoint in time (I),

[0103] (c) testing a sample of a body fluid from a patient suspected ofhaving a malignant tumour, said sample taken at a later point in time(II), with an antibody reagent specific for uPA:PAI-1 complexes underantibody binding conditions,

[0104] (d) determining the binding of the reagent to uPA:PAI-1 complexesin said sample of body fluid taken at said later point in time (II), and

[0105] (e) determining the difference between the level of saiduPA:PAI-1 complexes determined at said first point in time (I) with thevalue determined at said later point in time (II), and

[0106] (f) correlating said difference with an established level ofdifference which is indicative of a high likelihood of tumour presenceor metastasis.

[0107] In a preferred embodiment, the determination of the uPA:PAI-1complexes is performed by using an immunoassay, such as an ELISA or RIA,or by using an activity assay or other assays as described in furtherdetail above with regard to PAI-1 assays.

[0108] In another aspect, the invention relates to a method ofpredicting the prognosis of an individual subject having or suspected ofhaving a malignant; tumour, the method comprising

[0109] a) determining the level of uPA:PAI-1 complexes in malignant orpotentially malignant tissue or another sample, such as plasma, serum,or urine, from a number of subjects having or suspected of having amalignant tumour,

[0110] b) establishing a threshold level of uPA:PAI-1 complexes above orequal to which a value is indicative of a high likelihood of non-clinically evident tumour metastasis resulting in a poor prognosis,

[0111] c) correlating the level of uPA:PAI-1 complexes of the individualsubject with the value established in b) in order to determine theprognosis of the individual subject, and optionally

[0112] d) if the likelihood of a poor prognosis is high, allocating theindividual subject to subsequent antineoplastic treatment.

[0113] The invention also encompass a method of selecting a subjecthaving or suspected of having a malignant tumour for anti-PAI-1treatment, the method comprising

[0114] a) determining the level of uPA:PAI-1 complexes in malignant orpotentially malignant tissue or another sample, such as plasma, serum,or urine, from the subject,

[0115] b) correlating said value with an established threshold leveldetermined as described above, and

[0116] c) selecting for anti-PAI-1 treatment patients having a uPA:PAI-1complexes level above or equal to the predetermined threshold level.

BRIEF DESCRIPTION OF DRAWINGS

[0117] The invention will now be described in more detail with referenceto the drawings in which

[0118]FIG. 1 is a zymogram showing reverse zymography for plasminogenactivator inhibitor in culture fluid conditioned bydexamethasone-treated human fibrosarcoma cells of the line HT-1080 orumbilical cord endothelial cells before and after passage throughSepharose columns coupled with monoclonal antibodies againsttrinitrophenyl (control) and e-PAI,

[0119]FIG. 2 is a photography showing SDS-PAGE and reverse fibrinagarose zymography of HT-100 cell medium and e-PAI purified byimmunosorbent chromatography with a monoclonal antibody against e-PAI,

[0120]FIG. 3 is a graph showing neutralization of inhibitory action ofe-PAI by monoclonal antibody against e-PAI,

[0121]FIG. 4 is a zymogram showing binding of complexes of u-PA withe-PAI to Sepharose columns with monoclonal antibodies against e-PAI,

[0122]FIG. 5 is a photography showing immunoperoxidase staining ofHT-1080 cells with a monoclonal antibody against e-PAI,

[0123]FIG. 6 is a univariate analysis of tumour PAI-1 content in 57patients with colon adenocarcinoma,

[0124]FIG. 7A shows univariate survival curves of 293 patients withcolon cancer; patients were divided according to an optimized plasmaPAI-1 cut-off value (0.58 ng/mg protein); OS=overall survival,RR=relative risk, and the numbers indicate number of patients at risk,

[0125]FIG. 7B shows univariate survival curves of 316 patients withcolon cancer; patients were divided according to the optimized plasmacut-off value calculated from the first data set of 293 patients (FIG.7A); OS=overall survival, RR=relative risk, and the numbers indicatenumber of patients at risk,

[0126]FIG. 8A shows plasma uPA, uPAR and PAI-1 levels in a patient whodeveloped liver metastasis after surgical resection from colon cancer,

[0127]FIG. 8 shows plasma uPA, uPAR and PAI-1 levels in a patient whohad complete resection of her primary colon cancer and who did notexperience relapse,

[0128]FIG. 9 shows a uPA:PAI-1 complex ELISA measuring uPA:PAI-1standard (—) uPA (▪—▪) and PAI-1 (▴—▴), and

[0129]FIG. 10 shows the absorbance of a UPA:PAI-1 complex ELISAmeasuring uPA:PAI-1 complexes in 13 breast cancer cytosols.

MODES FOR CARRYING OUT THE INVENTION

[0130] The invention will be described in further detail with referenceto the examples. Examples 1 to illustrate the production and use ofmonoclonal antibodies against an endothelial type plasminogen activatorinhibitor (e-PAI) released into the culture fluid fromdexamethasone-treated human fibrosarcoma cells. The inhibitor inhibitshuman urokinase-type plasminogen activator (u-PA) and tissue-typeplasminogen activator (t-PA).

EXAMPLE 1

[0131] Production of the antigen used for immunization

[0132] Inhibitor was purified from serum-free conditioned culture fluidof dexamethasone-treated human fibrosarcoma cells of the line HT-1080(ATCC CCL121) by a procedure adapted from that described by van Mourik,J. A., Lawrence, D. A., and Loskutoff, D. J. (1984) J. Biol. Chem. 259,14914-14921 for the plasminogen activator inhibitor from bovineendothelial cells. The ET-1080 cell line was maintained as a monolayerculture, using Dulbecco-modified Eagle's medium supplemented with 10%fetal bovine serum. Serum-free culture fluid was prepared from confluentmonolayer cultures. Dexamethasone, a synthetic glucocorticoid, was addedto the serum-free cultures in a concentration of 10-6M. HT-1080 cellsproduce relatively high amounts of u-PA, which under the cultureconditions used is in the proenzyme form. Before purification ofinhibitor, the culture fluid was depleted of u-PA by passing it througha column of monoclonal anti-u-PA IgG immobilized on Sepharose (Nielsen,L. S., Hansen, J. G., Skriver, L., Wilson, E. L., Kaltoft, K., Zeuthen,J., and Danø, K. (1982) Biochemistry, 24, 6410-6415). The culture fluidwas then applied to a column of concanavalin A-Sepharose equilibratedwith 0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl (PBS), at a flow rateof 30 ml per h, using 5 ml Concanavalin A-Sepharose per liter culturefluid. The column was washed with 5 column volumes of PBS with 0.3 MNaCl. Bound protein was eluted with PBS with 0.5 M NaCl and 0.2 Ma-methyl-D-mannoside. Fractions containing the peak of protein, asdetermined by measuring the absorbancy at 280 nm, were pooled and usedfor further analysis.

[0133] From photometric scanning at 600 nm of Coomassie Blue stainedpolyacrylamide gels, the partly purified preparation was estimated tocontain approximately 75% of a Mr/54,000 protein, the electrophoreticmobility of which coincided with inhibitory activity as determined byreverse zymography (see below). Before immunization this preparation wasdialysed against PBS.

[0134] Immunization of BALB/c-mice

[0135] 4 BALB/c-mice were immunized intradermally with approximately 20μg of the Mr/54,000 protein obtained above in Freund's incompleteadjuvant on day 0, 7, 14, and 21. The plasma of each mouse was analyzedby ELISA (Enzyme Linked Immunosorbent Assay) and the mouse showing thehighest titer against the immunization preparation was chosen forintraveneous injection and fusion with myeloma cells. The intraveneousinjection of a similar dose as above dissolved in PBS was given on day28 and the spleen was removed 3 days later.

[0136] Cell fusion and culture of cells

[0137] Spleen cells were mixed with NSI-Ag 4/1 myeloma cells (resistantto 0.1 mM 6-thioguanine; synthesize but do not secrete kappa lightchains) (Köhler and Milstein (1976) Eur. J. Immunol. 6, 511-519) in aratio of 10:1 (108 spleen cells to 107 NSI-Ag 4/1 cells) and incubatedwith 1 ml of 50% (wt/vol) polyethylene glycol in a phosphate-bufferedsaline solution for 90 sec. at 37° C. Dulbecco's modified Eagle's medium(20 ml) was added to the suspension, and the cells were centrifuged at1000×g. The cell pellet was resuspended in 96 ml ofhypoxanthine/aminopterin/thymidine medium (Littlefield, J. W. (1964)Science 145, 709-710) supplemented with 10%fetal bovine serum and wasdistributed in 48 wells of Costar trays (Costar, Cambridge, Mass.). Themedium was changed twice weekly.

[0138] Selection of hybridomas

[0139] For screening of hybridoma supernatants using ELISA (EnzymeLinked Immunosorbent Assay), wells of Immuno Plates were coated with 100μl per well of concanavalin A-Sepharose-purified plasminogen activatorinhibitor containing/4 mg of protein per ml in 0.1 M Na2CO3, pH 9.8overnight at 37° C. In order to block residual binding sites, the wellswere incubated with 0.25% BSA in PBS for more than 15 minutes. Then thewells were incubated with hybridoma supernatants for 1 hour and finallywith peroxidase-conjugated rabbit antibodies against mouse Ig(Dakopatts, Copenhagen, Denmark), diluted 1:800 in PBS with 0.1% Tween20 for 1 hour. Peroxidase reaction was performed for 5 min. with 100 mlof 0.1% o-phenylenediamine, 0.01% H2O2 in 0.1 M citrate-phosphate, pH5.0. The reaction was stopped by the addition of 100 ml of 1 M H₂SO₄,and absorbancy was read at 492 nm.

[0140] For screening by immunoblotting, proteins in 10 ml of serum-freemedium from T-1080 cells were concentrated by precipitation withtrichloroacetic acid and separated by SDS-PAGE in a 10 cm wide lane. Theproteins were transferred electrophoretically (10 V, 250 mA for 16 h atroom temperature) from the polyacrylamide gel to nitrocellulose paper.The transfer buffer used was 0.125 M Tris HCl, 0.l M glycine, 20% (v/v)methanol, 0.1% (w/v) SDS, pH 8.6. The nitrocellulose paper was washed in0.05 M Tris HCl, pH 7.4, 0.15 M NaCl, 1% Triton X-100 (TBS-Triton) for15 min. at room temperature and incubated for 30 min. with TBS-Tritoncontaining human serum albumin (10 mg/ml). The paper was then washed2×15 min. in TBS-Triton. Vertical lanes were cut out and incubatedovernight at 4° C. with culture supernatants from the hybridomas. Thelanes were washed in TBS-Triton for 3×15 min., incubated for 1 h at roomtemperature with peroxidase-conjugated rabbit IgG anti-mouseimmunoglobulins (diluted 1:50 in TBS-Triton), and washed for 3×10 min.in 0.05 M Tris-HCl, pH 7,6. The peroxidase reaction was then performedwith 0.5 mg/ml of di-aminobenzidine in 0.001% H₂O₂ for 5 min. at roomtemperature.

[0141] As a control nitrocellulose lanes were incubated with supernatantfrom hybridomas (Hy 2.15) producing antibody of irrelevant specificity(anti-trinitrophenyl) (Shulman, M., Wilde, C. D., and Köhler, C. (1978)Nature, 276, 269).

[0142] After 10 days of cultivation of the hybridomas supernatant from16 primary wells snowed a strongly positive ELISA reaction. Thehybridomas from said wells were cloned and recloned by limiting dilution(Kennett, R. H., McKearn, J. T., and Bechtol, K. B. (1980) MonoclonalAntibodies. Hybridomas: A New Dimension in Biological Analysis (Plenum,New York)) . After cloning and recloning 4 stable ELISA-positive clonesremained. Immunoblotting analysis showed that all four clones producedantibody which reacted with an Mr/54,000 band in crude conditionedculture fluid from HT-1080 cells.

[0143] Purification of antibodies

[0144] Monoclonal antibodies produced by the 4 clones obtained werepurified from hybridoma culture fluid on a protein A-Sepharose column asfollows: 200 μl of conditioned culture fluid from hybridomas was appliedto a 5 ml protein A-Sepharose column (12×43 mm). The column was washedwith 30 ml of 0.1M Tris HCl, pH 8.1. Elution was performed with 0.1Msodium acetate, pH 4.0, 0.15 M NaCl. Fractions of 2 ml were collected intubes containing 200 μl of 1 M Tris-HCl, pH 9.0. The IgG concentrationin the purified preparation was determined by spectrophotometry at 280nm (A280 nm 1%=14). Concentrations of IgG in impure solutions weredetermined by single radial immunodiffusion using purified mouse IgG asa standard.

[0145] Characterisation of the antibodies produced by the clonedhybridomas

[0146] The classes and subclasses of the antibodies produced by thehybrid clones were analysed by immunodiffusion against class- andsubclass-specific goat antibodies (Meloy, V. A., USA). All 4 antibodiesproduced by the 4 clones were of the IgG₁ sub-class.

[0147] Isoelectric focusing of the 4 purified monoclonal antibodies inslab gels containing 6% polyacrylamide and 6carrier ampholyte solution(Pharmalyte) showed that their isoelectric points were different(ranging between 5 and 7.5). The binding characteristics of theantibodies to solid-phase inhibitor as measured by ELISA differedgreatly. They were therefore considered to originate from differenthybridization events. The four clones were designated anti-plasminogenactivator inhibitor clones 1, 2, 3, and 4, respectively.

[0148] Cross-reaction of antibodies against plasminogen activatorinhibitor from human fibrosarcoma cells with other plasminogen activatorinhibitors.

[0149] Conditioned culture fluid from human umbilical cord endothelialcells also contains a plasminogen activator inhibitor detectable byreverse fibrin-agarose zymography (Sprengers, E. O., Verheijen, J. H.,van Hindsberg, V. W. M., and Emeis, J. J. (1984) Biochim. Biophys. Acta.801, 163-170). This inhibitor has an electrophoretic mobilityindistinguishable from that of the HT-1080-inhibitor. FIG. 1 is azymogram showing reverse zymography for plasminogen activator inhibitorin culture fluid conditioned by HT-1080 cells (a-c) or umbilical cordendothelial cells (d-f) before (a, d) and after passage of Sepharosecolumns coupled with monoclonal antibodies against TNP (b, e) andagainst HT-1080 plasminogen activator inhibitor (c, e). For couplingprocedure see Example 2. Two one ml columns containing approximately 1mg of monoclonal anti-TNP IgG and monoclonal anti-plasminogen activatorinhibitor IgG from clone 1, respectively, were equilibrated with abuffer containing 0.1 M Tris HCl, pH 8.1, 0.1% Triton X-100. To bothcolumns was added 1 ml of serum-free cell culture fluid from HT-1080cells and the run-through was collected. Serum-free cell culture fluidfrom human umbilical cord endothelial cells was treated identically.After electrophoresis, the gel was processed for reverse zymography forplasminogen activator inhibitors with an incubation period of 1.5 hours.Reverse zymography was carried out as described by Eriksson, L. A.,Lawrence, D. A., and Loskutoff, D. J. (1984) Anal. Biochem. 137,454-463. Plasminogen activator inhibitors in SDS polyacrylamide gels aredetected by layering the gels over agarose gels containing fibrin,plasminogen, and plasminogen activator. Inhibitors diffuse into thefibrin/plasminogen/plasminogen activator gel from she polyacrylamidegel, and their presence is revealed by zones of fibrin resistant toplasminogen activator-catalyzed lysis. The position of Mr-markers areindicated.

[0150] Passage of HT-1080 medium through the Sepharose column withantibodies from anti-inhibitor IgG clone 1 removed the inhibitoryactivity as revealed by reverse fibrin-agarose zymography; there was noeffect of passage through a control column with a monoclonal controlantibody (anti-TNP IgG). When plasminogen activator inhibitor from saidhuman endothelial cells are applied to the column with monoclonalantibodies from clone 1 the inhibitor is bound to the column (FIG. 1).When columns with the antibodies from clones 2, 3, or 4 were used, theresults were identical (results not shown). This demonstratesimmunological similarities between this inhibitor and the HT-1080plasminogen activator inhibitor.

[0151] Using the same technique, it has also been shown that rabbitantibodies against the HT-1080-inhibitor cross-react with a plasminogenactivator inhibitor extracted from human blood platelets prepared by themethod described by Erikson, L. A., Ginsberg, M. H., and Loskutoff, D.J. (1984), J. Clin. Invest., 74, 1465-1472.

[0152] It has been reported that the endothelial cell plasminogenactivator inhibitor, the platelet inhibitor (Erikson, L. A., Ginsberg,M. H., and Loskutoff, D. J. (1984), J. Clin. Invest., 74, 1465-1472) andthe inhibitor from rat hepatoma cells of the HTC line show immunologicalsimilarities (D. J. Loskutoff and T. D. Gelehrter, personalcommunication). Thus it appears that the HT-1080-inhibitor is similar toa number of plasminogen activator inhibitors isolated from differentcells and tissues.

[0153] Immunosorbent purification of inhibitor

[0154] After coupling to Sepharose, antibody produced byanti-plasminogen activator inhibitor clone 1 was used for purificationof inhibitor from HT-1080 cell culture fluid by a column procedure. 8 mgof monoclonal antibodies from anti-inhibitor IgG clone 1 was coupled to2 ml of cyanogen bromide-activated Sepharose 4B. The material was packedin a column (20×16 mm), which was equilibrated with 0.1 M Tris HCl, pH8.1. Conditioned cell culture fluid from HT-1080 cells was applied at aflow rate of 50 ml/hr. The column was washed with equilibration buffer(flow rate 50 ml/hr) followed by 0.1 M Tris HCl, pH. 8.1, 1 M NaCl (flowrate 50 ml/hr). Elution was performed at a flow rate of 25 ml/h with 0.1M CH₃COOH, pH 2.7, collecting fractions of 2 ml into tubes containing200 ml of 1 M Tris HCl, pH 9.0 in order to neutralize the eluate.Fractions containing protein as determined by absorbance measurements at280 nm were pooled.

[0155] For Quantification of inhibitory activity 0.005 Ploug Unitsurokinase standard in 25 ml of assay buffer was mixed with 25 ml ofinhibitor diluted in the same buffer; this mixture was diluted to 500 mlwith assay buffer and added to [¹²⁵I]-fibrin plate wells (see Example3). From assays with fixed concentrations of urokinase standard andserial dilutions of inhibitor preparations, the dilution of inhibitorcausing 50% inhibition of the urokinase standard was calculated. Theamount of inhibitor in wells with 50% inhibition was defined as 0.0025inhibitor unit (Inh.U.). Before assay, inhibitor preparations weretreated with SDS to a final concentration of 0.1%, and after a one hourincubation at 25° C., Triton X-100 was added to a final concentration of1%.

[0156] Results from the purification of inhibitor from humanfibrosarcoma cells are shown in Table I. TABLE 1 Purification ofinhibitor from human fibrosarcoma cells by monoclonal antibody coupledto Sepharose Total Specific inhibitory inhibitory Volume Proteinactivity activity Fraction ml mg Inh.Units Inh.Units/mg Yield %Conditioned 740 59.9 19,200 321 100 culture fluid applied to column Runthrough 740 ND 2,600 ND 14 Wash 1 10 ND ND ND ND Wash 2 40 ND ND ND NDEluate 14.2 1.14 16,400 14,400 85

[0157] As shown in Table I, 86% of the inhibitory activity was bound bythe column. After washing 99% of the inhibitory activity bound to thecolumn could be eluted at low pH. A 45-fold purification of inhibitoryactivity was obtained and the eluate contained only an Mr/54,000 proteinband as evaluated by Coomassie Blue staining of a SDS-polyacrylamidegel. The electrophoretic mobility of this protein coincided with themobility of inhibitory activity as determined by reverse fibrin-agarosezymography (FIG. 2). The samples subjected to SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) were: 1.5 ml crude culture fluid from HT-1080cells (a), eluate corresponding to 10 mg of protein (b), 10 ml of crudeculture fluid from HT-1080 cells (a′), and eluate corresponding to 50 ngof protein (b′) After electrophoresis, the gels were either stained withCoomassie Blue (a, b), or inhibitor in the gels was visualized byreverse fibrin agarose zymography or 2 hours (a′, b′). The position ofthe following markers are indicated: Rabbit phosphorylase b (97 K) ,Bovine serum albumin (67 K), ovalbutin (43 K), carbonic anhydrase (30K), soybean trypsin inhibitor (20.1 K), and α-lactalbumin (14.4 K).

[0158] As judged from spectrophotometric scannings of stainedpolyacrylamide gels with crude culture fluid and the purifiedpreparation, the strong Mr/54,000 band was purified to the same extentas the inhibitory activity. SDS-PAGE in slab gels with a 6-16% linearconcentration gradient of polyacrylamide of crude culture fluid andrun-through from the immunosorbent column showed that the Mr/54,000 bandwas greatly diminished, while no other bands were affected (results notshown).

[0159] SDS-PAGE under reducing conditions showed one band withMr/54,000, indicating that the purified inhibitor consisted of onepolypeptide chain (results not shown).

EXAMPLE 3

[0160] Neutralization of inhibitory activity

[0161] The effect of the monoclonal antibodies on the inhibitoryactivity of the inhibitor was tested by a +1251(-fibrin plate assay,which involved the activation of plasminogen by u-PA and the subsequentdegradation of +1251(-fibrin by the plasmin formed (cf. Nielsen, L. S.,Hansen, J. G., Andreasen, P. A., Skriver, L., Danø, K., and Zeuthen, J.(1983) EMBO J., 2, 115-119). The +1251(-fibrin plate assay was carriedout as follows: 10 ng of inhibitor was added to +125I(-fibrin plateassay wells together with 0.2 ng active u-PA, 1 mg of Gluplasminogen andIgG as indicated, in a total volume of 500 ml of 0.1 M Tris HCl, pH 8.1,0.1% Triton X-100, 0.25% gelatine (assay buffer). radioactivity releasedin parallel control assays without u-PA (approximately 500 cpm) wassubtracted and the radioactivity released in the presence of inhibitorcalculated as a percentage of that released in the absence of inhibitor(approximately 3000 cpm). The total radioactivity in the +125I(-fibrinplate assay wells was approximately 60,000 cpm. Each point representsthe mean of two determinations. A neutralization of inhibitory activitythat increased with increasing concentrations of anti-inhibitor IgG fromclone 2 was observed (FIG. 3, ()) while there was no significant effecton inhibition of antibodies from clone 1 (FIG. 3 (o)) and the monoclonalcontrol antibody of irrelevant specificity (anti-TNP IgG) (FIG. 3 (A)).No neutralizing effect was observed with anti-inhibitor IgG from clone 3and 4 (results not shown).

EXAMPLE 4

[0162] Binding of uPA/inhibitor complexes to monoclonal anti-inhibitorantibodies

[0163] It has been shown that u-PA forms an equimolar complex with theHT-1080 inhibitor. This complex has an electrophoretic mobilitycorresponding to Mr/110,000 in SDS-PAGE and is detectable because itregains its plasminogen activator activity as measured by fibrin-agarosezymography: Plasminogen activator activity in poly acrylamide gels isdetected by layering the gels over agarose gels containing fibrin andplasminogen—the plasminogen activators diffuse into the agarose gels andactivate plasminogen to produce visible lysis zones (Granelli-Piperno,A. and Reich, E. (1978) J. Exp. Med., 148, 223-234). FIG. 4 is azymogram showing the binding of complexes of u-PA and HT-1080-inhibitorto Sepharose columns with monoclonal antibodies against the HT-1080inhibitor. One ml columns containing approximately 1 mg of monoclonalanti-TNP antibody (a), antibodies from anti-inhibitor IgG clone 1 (b),or monoclonal anti-plasminogen activator inhibitor IgG from clone 2 (c)were equilibrated with a buffer containing 0.1 M Tris HCl, pH 8.1, 0.1%Triton X-100, 0.25% gelatin. One ml u-PA-inhibitor complex obtained byincubating the activator (25 ng/ml) with the inhibitor (500 ng/ml) for 1h at 25° C. in a buffer of 0.1M Tris-HCl, pH 8.1, 0.1%. Triton X-100 wasadded to each column, and 75 ml of the run-through from each column wassubjected to SDS-PAGE followed by zymography for plasminogen activators.The positions of Mr-markers are indicated. Antibodies fromanti-inhibitor clone 2 bound these complexes, while no binding wasobserved with antibodies from anti-inhibitor clone 1 or anti-TNP.Likewise, monoclonal anti-plasminogen activator inhibitor IgG from clone4 bound complexes, while antibodies from 3 did not (results not shown).Said differential reactivities can be used in the quantitation of freeversus complex-bound inhibitor.

EXAMPLE 5

[0164] Immunocytochemical localization of the inhibitor

[0165] The monoclonal anti-plasminogen activator inhibitor antibodiescan be used for immunocytochemical localization of the inhibitor innormal or malignant cells and tissues.

[0166] HT-1080 cells cultured in serum free medium in the presence ofdexamethasone were seeded on microscope slides and fixed for 30 min.with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3. Afterwashing with 0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl (TBS) containing 1%Triton X-100 (TBS-Triton) for 30 min., the cells were exposed to 10%rabbit serum in TBS for 30 min, and incubated overnight at 4° C. withpurified monoclonal antibody (10 mg/ml) diluted in TBS with 10% rabbitserum. Following a 1 h temperature reequilibration, the cells werewashed with TBS-Triton and bound monoclonal antibody was demonstrated byincubation with peroxidase conjugated rabbit anti-mouse IgG (1:60)diluted in TBS with 10% rabbit-serum followed by development withdiaminobenzidine-hydrogen peroxide. The cells were lightlycounterstained with haematoxylin, dehydrated, mounted and photographed.A strong granular staining was observed often with a perinuclearlocalization together with a weak diffuse staining apparentlydistributed in the entire cytoplasm (FIG. 5, top). A distinct granularstaining was observed using antibody from all 4 clones (results shownfor clone 1 only). When the monoclonal anti-inhibitor antibody wassubstituted by monoclonal IgG of irrelevant specificity (anti-TNPantibody) (FIG. 5, bottom) or by buffer alone, no staining was seen.

[0167] The invention has been illustrated with reference to theproduction and use of monoclonal antibodies against a Mr/54,000plasminogen activator inhibitor released into the culture fluid fromdexamethasone-treated human fibrosarcoma cells, but since said inhibitoris immunologically similar to the plasminogen activator inhibitorsderived from human endothelial cells, human platelets and rat hepatomacells, it should be understood that monoclonal antibodies againstplasminogen activator inhibitors from these sources and plasminogenactivator inhibitors which are immunologically similar to theplasminogen activator inhibitors from any of these sources also fallwithin the scope of the invention.

EXAMPLE 6

[0168] Prognostic value of plasma PAI-1 in patients with colorectalcancer

[0169] All studies published until now on the prognostic value of PAI-1in cancer are based on determinations in tumour extracts. For most typesof cancer, the development of more effective diagnostic methods hasresulted in earlier detection and thus smaller tumour size at the timeof surgery. This makes it increasingly difficult to gain access tofrozen, unfixed tumour samples. If similar prognostic information asthat obtained by analyzing tumour extracts could be obtained by apreoperatively collected plasma sample, it would significantly increaseits clinical value with sample collection being easier and lessinvasive.

[0170] Colon cancer affects one out of twenty in the U.S. and in mostWesternized countries with more than 155,000 new cases diagnosed eachyear, this disease accounts for 15% of all cancers and constitutes amajor public health problem. The disease is divided according to Dukes'stage A-D. Recently, adjuvant chemotherapeutic treatment of patientswith Dukes' C has been recommended. However, a large fraction of thesepatients, who are cured by the primary surgical treatment, will stillreceive chemotherapy. A reliable means of selecting those patients athighest risk of recurrence would allow for adjuvant therapy to belimited to this group and thus spare a large number of patients from theoften severe side effects associated with chemotherapy.

[0171] MATERIALS AND METHODS

[0172] Patients

[0173] The patients from whom the plasma samples were drawn all hadsurgery for colorectal cancer. Patients were followed regularly in theout-patient department. Survival data were obtained from the CentralDanish Registry of Death.

[0174] Plasma sampling

[0175] Plasma was obtained from 609 patients with colorectal cancer Theplasma was collected preoperatively and stored at −80° C. until analyzedfor PAI-1 content.

[0176] PAI-1 ELISA

[0177] PAI-1 was determined using a sandwich ELISA (Grøndahl-Hansen etal., 1993, “High levels of urokinase-type plasminogen activator (u-PA)and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas areassociated with poor prognosis”; Cancer Research 53, 1513-1521) withmonoclonal catching and detecting antibodies. As catching antibody wasused PAI-1 monoclonal antibody clone 1, and as detecting antibody wasused PAI-1 monoclonal antibody clone 7. This assay detects both latentand active PAI-1 and in addition recognizes PAI-1 bound to uPA and tPA(unpublished results, J. Grøndahl-Hansen). PAI-1 was measured as ng/mgprotein.

[0178] Statistical methods

[0179] The patients were randomly divided into two groups, each of whichbeing representative for the total number of patients. The first groupof patients (29:3 patients) were used to search for an optimum cut-offvalue to separate patients into two groups with different survival. Thisoptimized cut-point was then tested in the second group of patients (316patients).

[0180] Results

[0181] The optimized plasma PAI-1 cut-off value was calculated to be0.58 ng/mg protein. Using this cut-off point in the 293 patients gave arelative hazard rate of 1.5, i.e. patients with plasma PAI-1 levelsabove this cut-off point had a 50% higher risk of death than patientswith plasma PAI-1 levels below the cut-off point (FIG. 7A).

[0182] Applying the optimized cut-off point on the group of 316 patientsgave identical results (FIG. 7B).

[0183] DISCUSSION

[0184] This study of the prognostic value of preoperative plasma PAI-1in patients undergoing surgical resection for colorectal cancer suggeststhat high plasma levels of PAI-1 are associated with short overallsurvival. Measurement of plasma PAI-1 might then be used to dividecolorectal cancer patients into groups of low versus high risk ofrecurrence. Only patients at high risk of recurrence should then beoffered adjuvant systemic chemotherapy. The present study is the firstof its kind, and the prognostic value of plasma PAI-1 in other types ofcancer can and should be evaluated in a similar manner.

EXAMPLE 7

[0185] Predictive test of plasma PAI-1 measurements in patients havingcolon adenocarcinomas

[0186] In this example is described a predictive test to identifypatients who should be offered anti-PAI-1 therapy.

[0187] MATERIALS AND METHODS

[0188] Patients

[0189] Patients with colon adenocarcinoma Dukes' B+C referred foradjuvant therapy subsequent to radical resection of their tumours.

[0190] PAI-2 ELISA

[0191] Plasma samples from the patients with colon adenocarcinoma isstored at −80° C.

[0192] PAI-1 is determined using a sandwich ELISA (Grøndahl-Hansen etal., 1993) with monoclonal catching and detecting antibodies. Ascatching antibody is used PA-1 monoclonal antibody clone 1 and asdetecting antibody is used PAI-1 monoclonal antibody clone 7 (WO87/00549). This assay detects both latent and active PAI-1, and is inaddition recognizing PAI-1 bound to uPA and tPA (unpublished results, J.Grøndahl-Hansen). PAI-1 is measured in interim units by calibration withstandard preparations obtained from The National Institute forBiological Standards and Control, Hertfordshire, UK. The intra- andintervariations for both assays are below 11%.

[0193] TREATMENT

[0194] The patients are divided into groups on the basis of their tumouror plasma PAI-1 content. All patients in the groups having a high tumourand/or plasma PAI-1 content will receive anti-PAI-1 treatment. Clinicalresponses will be recorded according to standard procedures (EORTC).Patients will be post-stratified according to tumour and/or plasma PAI-1content (patients with tumour PAI-1 levels at or above versus below theestablished median value of 0.775 InterimU/mg protein and patients withplasma PAI-1 levels on or above the established median value of 0.58ng/mg protein in serum) and number and duration of objective responsesas well as survival will be compared between groups of patients.

EXAMPLE 8

[0195] Diagnostic value of plasma PAI-1 in patients with colorectalcancer

[0196] Follow-up of cancer patients in either surgically or medicallyinduced remission most often involves only clinical examination. Withthe high recurrence rate in many cancer types, a sensitive diagnosticassay which is capable, with high sensitivity, of identifyingnon-clinically evident recurrence will be of significant value. Such atest might also be useful in screening high-risk populations for theoccurrence of cancer.

[0197] MATERIALS AND METHODS

[0198] Patients

[0199] The patients from whom the plasma samples were collected all hadsurgery for colorectal cancer. Patients were followed regularly in theout-patient department.

[0200] Plasma sampling

[0201] Plasma was obtained preoperatively, peroperatively, on days 2 and7 postoperatively and then every three months until clinical relapse wasevident.

[0202] PAI-1 ELISA

[0203] The PAI-1 ELISA was performed as described above in Example 6.

[0204] Results

[0205]FIG. 8A shows plasma uPA, uPAR and PAI-1 levels in a patient withprogressive colorectal cancer. At the time of surgery, the patient hadno signs of disseminated disease. The preoperative plasma PAI-1 levelwas elevated, but decreased as a result of surgical removal of theprimary tumour. However, at 3 months follow-up, PAI-1, had increased 3times over the preoperative plasma PAI-1 value, and the patient nowpresented clinically evident liver metastasis. FIG. 8B shows plasma uPA,uPAR and PAI-1 from a patient who had her primary colon cancer removedand who did not experience a relapse of her cancer. The preoperativeplasma PAI-1 value was elevated, but decreased subsequent to surgery.During the 3-month follow-up period, no increase in plasma PAI-1 wasobserved, which is consistent with no appearance of metastasis.

[0206] DISCUSSION

[0207] This study on patients with colorectal cancer suggests thatmeasurement of plasma PAI-1 might be used as a marker for relapse of thecancer disease. Routine measurement of plasma PAI-1 might then be usedto follow patients who have achieved a surgically or medically inducedcomplete remission of their colorectal cancer. In many cancer diseases,early detection of a relapse is a prerequisite for a subsequent curativetherapy. Therefore, the diagnostic value of plasma PAI-1 in other typesof cancer should be evaluated in a similar manner.

EXAMPLE 9

[0208] Determination of uPA:PAI-1 complexes in patients

[0209] MATERIALS AND METHODS

[0210] The patients from whom the tumour cytosols were obtained all hadsurgery for breast cancer. Breast cancer cytosols were preparedaccording to EORTC guidelines.

[0211] uPA:PAI-1 ELISA

[0212] uPA:PAI-1 complexes were determined using a sandwich ELISA with apolyclonal uPA antibody as catching antibody and a monoclonal PAI-1antibody as detecting antibody. A uPA:PAI-1 standard was prepared fromHT-1080 sarcoma cell affinity purified PAT-1 activated with 4M guanidiumHCl and incubated with human active uPA (Serono). The sensitivity of theELISA is 1.5 ng/ml uPA:PAI-1 complexes (FIG. 9). No reactivity was foundwith pro-uPA, active uPA, latent PAI-1, active PAI-1 or active uPAincubated with latent PAI-1 (FIG. 9).

[0213] Results

[0214] By measuring with the above-mentioned ELISA, uPA:PAI-1 complexesin breast cancer cytosols obtained from 13 individual breast cancerpatients, immunoreactivity could be detected in dual cytosols wereobserved (FIG. 10).

1. A screening method for better ascertaining at least one variableselected from the group consisting of (1) the likelihood of the presenceof a tumor in a subject suspected of having a tumor, (2) the likelihoodof the presence of a malignant tumor in a subject suspected of having amalignant tumor (3) the likelihood of the presence of a tumor metastasisin a subject suspected of having a tumor metastasis, and (4) the likelyprogression of a malignant tumor in a subject, the method comprising (a)determining the level of a marker, selected from the group consisting of(i) PAI-1 protein abundance, (ii) uPA:PAI complex abundance (iii) thechange in PAI-1 protein abundance over time, and (iv) the change inuPA:PAI complex abundance over time, in a test sample, and, in the caseof (iii) and (iv) above, over a plurality of test samples taken atdifferent times, said test sample or samples being of or derived from abody fluid or tissue of said subject, said tumor being of a kind suchthat there is a correlation between the level of said marker and atleast one of variables (1)-(4) above, and (b) correlating the level ofsaid marker for said sample or samples, and the reference level of saidmarker for a corresponding reference sample or samples, whereby at leastone of variables (1)-(4) is better ascertained.
 2. The method of claim 1where the variable (1) is ascertained.
 3. The method of claim 1 wherethe variable (2) is ascertained.
 4. The method of claim 1 where thevariable (3) is ascertained.
 5. The method of claim 1 where the variable(4) is ascertained.
 6. The method of claim 1 where the marker is (i). 7.The method of claim 1 where the marker is (ii).
 8. The method of claim 1where the marker is (iii).
 9. The method of claim 1 where the marker is(iv).
 10. The method of claim 1 where the tumor is a breast tumor. 11.The method of claim 1 where the tumor is a gastric tumor.
 12. The methodof claim 1 where the tumor is a colon tumor.
 13. The method of claim 1where the tumor is a lung tumor.
 14. The method of claim 1 where thetumor is an ovarian tumor.
 15. The method of claim 14 where the markeris (i) and the variable is (1).
 16. The method of claim 1 where themarker is (i), the variable is (4), and the tumor is selected from thegroup consisting of breast, gastric, colon, and lung cancers.
 17. Themethod of claim 1 in which the sample is a frozen, unfixed tumor tissuesample or extract thereof.
 18. The method of claim 17 where the markeris (i) and (4) is better ascertained.
 19. The method of claim 18 wherethe tumor is selected from the group consisting of breast, gastric,colon, lung, and ovarian cancer.
 20. The method of claim 1 in which thesample is a body fluid.
 21. The method of claim 17 in which the sampleis plasma.
 22. The method of claim 21 in which the marker is (i) and thetumor is a pancreatic, ovarian, urinary tract, colon, colorectal orbreast tumor.
 23. The method of claim 21 in which the marker is (iii),and (4) is ascertained.
 24. The method of claim 23 in which a firstsample is taken preoperatively and a later sample is taken postoperatively.
 25. The method of claim 24 in which the tumor is a colontumor.
 26. The method of claim 7 in which the tumor is a breast tumor,and the sample is a cytosol.
 27. The method of claim 1 in which (3)applies, and the method determines whether there is more than a 40% riskof tumor metastasis within about five years.
 28. The method of claim 1in which (4) applies, and the method determines whether the subject hasat least a 50% higher risk of death.
 29. The method of claim 1 wheresaid level is determined by an immunoassay.
 30. The method of claim 29where said level is determined by a binding assay in which one of theassay reagents is a monoclonal antibody which binds a human endothelialtype plasminogen activator inhibitor produced by dexamethasone-treatedhuman HT-1080 fibrosarcoma cells (ATCC CCL121).
 31. The method of claim30 where said marker is (iii) or (iv) and said antibody further binds toa complex comprising urokinase-type plasminogen activator and ePAI. 32.The method of claim 30 where said antibody binds specifically to anantigenic determinant of human ePAI which is also specifically bound bythe monoclonal antibody secreted by the hybridoma of clone
 2. 33. Themethod of claim 31 where said antibody binds specifically to anantigenic determinant of human ePAI which is also specifically bound bythe monoclonal antibody secreted by the hybridoma of clone
 4. 34. Themethod of claim 1 where the malignant tumor is selected from the groupconsisting of mammary carcinomas, urological carcinomas, gynecologicalcarcinomas, non-small cell lung tumors, gastrointestinal cancers, braintumors, sarcomas, haematological malignancy and skin cancers.
 35. Themethod of claim 1 which further comprises monitoring the progression ofthe tumor in a patient by determining the level of the marker at aplurality of different times and correlating said levels with the timesof determination.
 36. A method of detecting the presence or predictingthe progression of a malignant tumor in a subject having or suspected ofhaving a malignant tumor, the method comprising (a) determining at afirst point in time (I) one or more of PAI-1 DNA abundance, PAI-1 mRNAabundance, or PAI-1 protein abundance in tumor tissue or a sample of abody fluid such as plasma, serum or urine from said subject, (b)determining at a later point in time (II) one or more of PAI-1 DNAabundance, PAI-1 mRNA abundance, or PAI-1 protein abundance in a sampleof body fluid from said subject, (c) determining the difference betweenthe abundance of said PAI-1 DNA, PAI-1 mRNA, or PAI-1 protein determinedat said first point in time (I) with the value determined at said laterpoint in time (II), and (d) correlating said difference with anestablished level of difference which is indicative of a high likelihoodof tumor presence or metastasis.
 37. A method according to claim 36wherein said first point in time (I) is preoperatively.
 38. A methodaccording to claim 36 wherein said second point in time (II) is at least2 weeks postoperatively.
 39. A method according to claim 36, wherein themalignant tumor is selected from the group consisting of mammarycarcinomas, urological carcinomas, gynaecological carcinomas, non-smallcell lung tumors, gastrointestinal cancers, brain tumors, sarcomas,haematological malignancy and skin cancers.
 40. A method of predictingthe presence or progression of a malignant tumor in a subject having orsuspected of having a malignant tumor, the method comprising (a)determining at a first point in time (I) uPA:PAI-1 complexes in a sampleof a tissue or a body fluid from said subject, (b) determining at alater point in time (II) uPA:PAI-1 complexes in a sample of a tissue orbody fluid from said subject, (c) determining the difference between theabundance of said uPA:PAI-1 complexes determined at said first point intime (I) with the abundance determined at said later point in time (II),and (d) correlating said difference with an established level ofdifference which is indicative of high likelihood of tumor presence ormetastasis.
 41. A method according to claim 40 comprising the steps of:(a) testing a tissue section from a malignant tumor or a sample of abody fluid from a patient having or suspected of having a malignanttumor, said sample taken at a first point in time (I), with an antibodyreagent specific for uPA:PAI-1 complexes under antibody bindingconditions, (b) determining the binding of the reagent to uPAP:PAI-1complexes in said tissue section or sample of a body fluid taken at saidfirst point in time (I), (c) testing a sample of a tissue or a bodyfluid from a patient having or suspected of having a malignant tumor,said sample taken at a later point in time (II), with an antibodyreagent specific for uPA:PAI-1 complexes under antibody bindingconditions, (d) determining the binding of the reagent to uPA:PAI-1complexes in said sample taken at said later point in time (II), (e)determining the difference between the level of said uPA:PAI-1 complexesdetermined at said first point in time (I) with the value determined atsaid later point in time (II), and (f) correlating said difference withan established level of difference which is indicative of a highlikelihood of tumor presence or metastasis.
 42. A method according toclaim 40, wherein the determination of the uPA:PAI-1 complexes isperformed by using an immuno-assay, such as an ELISA or RIA, or by usingan activity assay.
 43. A method of predicting the prognosis of anindividual subject having or suspected of having a malignant tumor, themethod comprising (a) determining the level of uPA:PAI-1 complexes inmalignant or potentially malignant tissue or body fluid from a number ofsubjects having a malignant tumor, (b) establishing a threshold level ofuPA:PAI-1 complexes above or equal to which a value is indicative of ahigh likelihood of non-clinically evident tumor metastasis resulting ina poor prognosis, (c) correlating the level of uPA:PAI-1 complexes ofthe individual subject with the value established in (b) in order todetermine the prognosis of the individual subject, and optionally (d) ifthe likelihood of a poor prognosis is high, allocating the individualsubject to subsequent antineoplastic treatment.
 44. A method ofdetecting the uPA:PAI-1 complex in a sample, said sample selected fromthe group consisting of body fluids, normal cells, malignant cells, andtissues, said method comprising detecting the antigen comprising theuPA:PAI-1 complex by incubating the sample with one or more antibodieswhich specifically bind PAI-1 in the uPA:PAI-1 complex to form anantibody/antigen complex, and detecting the antibody/antigen complex.45. The method of claim 36 wherein PAI-1 protein abundance is determinedin a sample of a body fluid.
 46. A method according to claim 44 whereinthe sample is a body fluid, and said body fluid is plasma.
 47. A methodaccording to claim 44 wherein the body fluid, normal or malignant cells,or other biological material is taken from a person suspected of havingbreast cancer.
 48. A method according to claim 44 wherein the sample isa tissue.
 49. A method of assaying the uPA:PAI-1 complex in a sample,said sample selected from the group consisting of body fluids, normalcells, malignant cells, and tissues, said method comprising assaying theantigen comprising the uPA:PAI-1 complex by incubating the sample withone or more antibodies which specifically bind PAI-1 in the uPA:PAI-1complex to form an antibody/antigen complex, and assaying theantibody/antigen complex.
 50. The method of 49, further comprisingassaying the total amount of uPA in said sample.
 51. A method ofpredicting the presence or progression of a malignant tumor in a subjecthaving or suspected of having a malignant tumor, the method comprising(a) determining preoperatively PAI-1 protein abundance in a plasmasample from said subject (b) determining at least two weekspostoperatively PAI-1 protein abundance in a plasma sample from saidsubject, (c) determining the difference between the abundance of saidPAI-1 protein determined preoperatively with the value determined atleast two weeks postoperatively, and (d) correlating said differencewith an established level of difference which is indicative of a highlikelihood of tumor presence or metastasis.
 52. A method according toclaim 51 wherein the malignant tumor is a colon tumor.
 53. A method ofdetecting the presence of a tumor in a subject suspected of having atumor, the method comprising (a) determining at a first point in time(I) PAI-1 protein abundance in tumor tissue or a sample of a body fluidfrom said subject, (b) determining at a later point in time (II) PAI-1protein abundance in a sample of body fluid from said subject, (c)determining the difference between the abundance of said PAI-1 proteindetermined at said first point in time (I) with the value determined atsaid later point in time (II), and (d) correlating said difference withan established level of difference which is indicative of a highlikelihood of tumor presence.
 54. A method according to claim 53,wherein the malignant tumor is selected from the group consisting ofmammary carcinomas, urological carcinomas, gynaecological carcinomas,non-small cell lung tumors, gastrointestinal cancers, brain tumors,sarcomas, haematological malignancy and skin cancers.
 55. The method ofclaim 54 wherein PAI-1 protein abundance is determined in a sample of abody fluid.
 56. The method of claim 2 where the marker is (ii).
 57. Themethod of claim 3 where the marker is (ii).
 58. The method of claim 4where the marker is (ii).
 59. The method of claim 5 where the marker is(ii).
 60. The method of claim 2 where the marker is (iv).
 61. The methodof claim 3, where the marker is (iv).
 62. The method of claim 4 wherethe marker is (iv).
 63. The method of claim 5 where the marker is (iv).64. The method of claim 7 where the marker is determined using asandwich ELISA.
 65. The method of claim 64 in which, in said assay, thePAI-1 is bound by an anti-PAI-1 monoclonal antibody.
 66. The method ofclaim 65 in which said antibody is one which binds specifically to anantigenic determinant by human PAI-1 which is also specifically bound bya monoclonal antibody selected from the group consisting of thosesecreted by hybridoma clones 1-4.
 67. The method of claim 66 in whichthe antibody is selected from the group consisting of the monoclonalantibodies secreted by hybridoma clones 1-4.
 68. The method of claim 7in which the complex is bound by an antibody which binds specifically toan antigenic determinant of human PAI-1 which is also specifically boundby the monoclonal antibody secreted by the hybridoma of clone 4 (ECACC00112120).
 69. The method of claim 68 in which the antibody is themonoclonal antibody secreted by the hybridoma of clone 4.